One of the most popular assays for detecting superoxide in cells and tissues involves the use of fluorescence-based techniques (Rothe, G. and Valet G. J. Leukoc. Biol. 47:440–448, 1990; Carter, W. et al. J. Leukoc. Biol. 55:253–258, 1994; Bindokas, V. et al. J. Neurosci. 16:1324–1336, 1996; Budd, S. et al. FEBS Lett. 415:21–24, 1997; Castilho, R. et al. J. Neurochem. 72:1394–1401, 1999; and Suzuki, H. et al. Hypertension 25:1083–1089, 1995). Generally, the red fluorescence arising from oxidation of hydroethidine (HE) (also called dihydroethidium (DHE), FIG. 1) is detected (Nakane, H. et al. Hypertension 35:595–601, 2000; Miller, F. et al. Circ. Res. 82:1298–1305, 1998; Kawase, M. et al. Stroke 30:1962–1968, 1999; Kim, G. et al. Stroke 33:809–815, 2002; Sorescu, D. et al. Circulation 105:1429–1435, 2002; Dantas, A. P. et al. Hypertension 39:405–411, 2002; Paravicini, T. et al. Circ. Res. 91:54–61, 2002; and Luo, J. et al. J. Neurosci. Meth. 120:105–112, 2002). HE is synthesized from sodium borohydride reduction of ethidium (E+), a two-electron oxidation product (Thomas, G. and Roques, B. FEBS Lett. 26:169–175, 1972). It is the common belief in the art that the reaction between superoxide and HE results in the formation of a two-electron oxidized product, E+, that binds to DNA and leads to the enhancement of red fluorescence (excitation, 500–530 nm; emission, 590–620 nm). Most previous fluorescence measurements have been performed using a kinetic mode and typically acquired at a single wavelength corresponding to that of E+ (Schuchmann, S. and Heinemann, U. Free Radic. Biol. Med. 28:235–250, 2000; Zou, A.-P. et al. Hypertension 37:547–553, 2001; Kevin, L. et al. Am. J. Physiol. Heart Circ. Physiol. 284: H566–H574, 2003; Benov, L. et al. Free Radic. Biol. Med. 25:826–831, 1998; and Vanden Hoek, T. et al. J. Mol. Cell Cardiol. 29:2571–2583, 1997). Alternatively, the red fluorescence due to oxidized HE was visualized in cells and tissues using fluorescence or confocal microscopy. This red fluorescence, often referred to as the “E+ fluorescence,” is inhibited by intracellular superoxide dismutase and other superoxide scavengers (Nakane, H. et al. Hypertension 35:595–601, 2000; Miller, F. et al. Circ. Res. 82:1298–1305, 1998; Kawase, M. et al. Stroke 30:1962–1968, 1999; Kim, G. et al. Stroke 33:809–815, 2002; Sorescu, D. et al. Circulation 105:1429–1435, 2002; Dantas, A. P. et al. Hypertension 39:405–411, 2002; Paravicini, T. et al. Circ. Res. 91:54–61, 2002; and Luo, J. et al. J. Neurosci. Meth. 120:105–112, 2002).
To the inventors' knowledge, the question of whether superoxide-dependent oxidation of HE actually generates E+ as a product has never been definitively answered. The answer to this question is important for the accurate detection and quantitation of superoxide through the use of HE.